PO
Undergraduate and Graduate Category: Physical and Life Sciences Degree Level: Bachelor of Science Abstract ID #340
Young but Immuno-‐competent: Phenoloxidase Ac?vity in Termite Eggs and Larvae
Abstract
AcCvaCon of the phenoloxidase (PO) proteolyCc cascade is a mechanism by which insects cope with infecCous agents. The quanCficaCon of the enzyme phenoloxidase can serve as a proxy for esCmaCng potenCal immune capabiliCes in an insect. PO exists in both an acCve (PO) and an inacCve form (pro-‐PO), and the amount of both can differ as a funcCon of the insect’s size, age, gender and immunological state. The dampwood termite Zootermopsis angus/collis is a eusocial insect that thrives in a wet, dark wooden environment. These feeding/nesCng condiCons also foster the development of a variety of microbes, posing significant pathogenic threats to colonies. While amounts of PO have been measured in termites, no study has ever aWempted to quanCfy PO in insect eggs. Researchers have dismissed eggs as too small and/or too immature to exhibit PO acCvity. Here, we demonstrate, for the first Cme, that pro-‐PO and PO are present in termite eggs, albeit in small quanCCes. We also demonstrate that the expression of the inacCve and acCve forms vary as the embryo progresses through its development: older embryos have higher levels of PO than recently ferClized eggs. Whether the levels of PO translate into higher resistance to infecCon remains to be tested. Yet, the presence of these enzymes in the most immature stage of the life cycle of a termite point to the role that pathogens play in the evoluCon and ontogeny of mechanisms of disease resistance.
Suzanne Kent, Erin Cole, and Rebeca Rosengaus
Results
Total PO AcCvity
Figure 5: PO ac?vity in worker termites compared to the larvae and eggs. The level of PO in eggs and larvae in negligible compared to that of workers
Figure 1: King (leJ) and queen (right) termites
IntroducCon
• Insects, including Zootermopsis angus/collis (Fig.1) have an immune system that protects them against foreign microbial invasion. • MelanizaCon, the pathway of melanin formaCon (Fig.2), and encapsulaCon of pathogens depends on PO and plays a crucial role in insect immunity • PO=proxy to measure potenCal immune capabiliCes of an insect • Z. angus/collis thrives in wet, dark wooden environments which fosters the growth of microbes. • PO has never been measured in the embryo stage of Zootermopsis
Figure 8: Three stages of termite eggs and 1st instar larvae 0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 108 114 120
Conclusion
Time (mins)
Total PO AcCvity Figure 2: The melaniza?on pathway that takes place in insects. Prophenoloxidase (ProPO) is cleaved in order to become the ac?ve form, pheloxidase (PO)
Figure 6: Young larvae contain ~10 ?mes more total PO ac?vity than the eggs. The total PO is also ~10 ?mes the amount of the ac?ve circula?ng PO, see Figure 7.
Materials and Methods
• Mature colonies of Z. angus/collis were collected from the Redwood East Bay Regional Park in California and maintained at Northeastern University • Eggs were obtained from these colonies or young colonies founded in the lab (Fig.3). Eggs and larvae in PBS were sonicated to break open the Cssue. • 50 μL of a sonicated sample were placed in a microplate well with either 70 μL of chymotrypsin (total PO) or PBS (acCve PO) and with 150 μL of an L-‐ dopa soluCon were added as a substrate for the PO that ulCmately generated melanin. • The plate was placed in a Synergy HT-‐I spectrophotometer, and the absorbance was measured every two minutes at 492 nm for a total of two hours . Samples turn brown in direct proporCon to the amount of PO in them (Fig. 4).
0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 108 114 120
Time (mins)
AcCve (CirculaCng) PO AcCvity
Figure 3: Pile of termite eggs and young incipient colony where eggs and young larvae are collected from
Figure 7: Circula?ng PO is much lower than total PO. The young larvae have ~10 ?mes more PO than the eggs. Yet even Stage 2 and 3 eggs (Fig. 8) had detectable levels of ac?ve PO.
• Workers have approximately 100x more total PO acCvity than eggs per microliter and ~10x more than young larvae. • The larvae have approximately 10x more total PO acCvity than eggs per microliter • Such differences in PO acCvity are likely due to differences in the expression level of PO-‐related genes in termites as they mature • Stage 1 egg results are not shown in the results because their data produced many outliers • This inconsistent data could be caused by the vitogellin bubbles as seen in Fig. 8 • This differenCal expression of PO has never before been examined or quanCfied in such young insects • • •
Figure 4: Micro plate with samples. The darker color represents the melanin. The darker rows beneath the lighter rows represent samples that contain chymotrypsin.
References
Rosengaus, R.B., Traniello, J.F.A. 2001. Disease suscepCbility and the adapCve nature of colony demography in the dampwood termite Zootermopsis angus/collis. Behavioral Ecology and Sociobiology, 50:546–556. Tsakas, S., Marmaras, V.J. 2010. Insect immunity and its signaling: an overview. ISJ, 7: 228-‐238. Vilmos, P., Kurucz, E., 1998. Insect immunity: evoluConary roots of the mammalian innate immune system. Immunology LeWers 62: 59-‐66.
Acknowledgements
0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 108 114 120
Time (mins)
Theo Bowe, Natalie Grace Schulz, John Paul ThoWam, Lyndie Parker, Esha Parikh, Grace Lee, Maeve Tobin, Sam Bosch. We thank the Godoy lab and Ryan Benson for the use of lab equipment and the Epstein lab for the use of the sonicator.
Young but Immuno-‐competent: Phenoloxidase Ac?vity in Termite Eggs and Larvae
Abstract
AcCvaCon of the phenoloxidase (PO) proteolyCc cascade is a mechanism by which insects cope with infecCous agents. The quanCficaCon of the enzyme phenoloxidase can serve as a proxy for esCmaCng potenCal immune capabiliCes in an insect. PO exists in both an acCve (PO) and an inacCve form (pro-‐PO), and the amount of both can differ as a funcCon of the insect’s size, age, gender and immunological state. The dampwood termite Zootermopsis angus/collis is a eusocial insect that thrives in a wet, dark wooden environment. These feeding/nesCng condiCons also foster the development of a variety of microbes, posing significant pathogenic threats to colonies. While amounts of PO have been measured in termites, no study has ever aWempted to quanCfy PO in insect eggs. Researchers have dismissed eggs as too small and/or too immature to exhibit PO acCvity. Here, we demonstrate, for the first Cme, that pro-‐PO and PO are present in termite eggs, albeit in small quanCCes. We also demonstrate that the expression of the inacCve and acCve forms vary as the embryo progresses through its development: older embryos have higher levels of PO than recently ferClized eggs. Whether the levels of PO translate into higher resistance to infecCon remains to be tested. Yet, the presence of these enzymes in the most immature stage of the life cycle of a termite point to the role that pathogens play in the evoluCon and ontogeny of mechanisms of disease resistance.
Suzanne Kent, Erin Cole, and Rebeca Rosengaus
Results
Total PO AcCvity
Figure 5: PO ac?vity in worker termites compared to the larvae and eggs. The level of PO in eggs and larvae in negligible compared to that of workers
Figure 1: King (leJ) and queen (right) termites
IntroducCon
• Insects, including Zootermopsis angus/collis (Fig.1) have an immune system that protects them against foreign microbial invasion. • MelanizaCon, the pathway of melanin formaCon (Fig.2), and encapsulaCon of pathogens depends on PO and plays a crucial role in insect immunity • PO=proxy to measure potenCal immune capabiliCes of an insect • Z. angus/collis thrives in wet, dark wooden environments which fosters the growth of microbes. • PO has never been measured in the embryo stage of Zootermopsis
Figure 8: Three stages of termite eggs and 1st instar larvae 0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 108 114 120
Conclusion
Time (mins)
Total PO AcCvity Figure 2: The melaniza?on pathway that takes place in insects. Prophenoloxidase (ProPO) is cleaved in order to become the ac?ve form, pheloxidase (PO)
Figure 6: Young larvae contain ~10 ?mes more total PO ac?vity than the eggs. The total PO is also ~10 ?mes the amount of the ac?ve circula?ng PO, see Figure 7.
Materials and Methods
• Mature colonies of Z. angus/collis were collected from the Redwood East Bay Regional Park in California and maintained at Northeastern University • Eggs were obtained from these colonies or young colonies founded in the lab (Fig.3). Eggs and larvae in PBS were sonicated to break open the Cssue. • 50 μL of a sonicated sample were placed in a microplate well with either 70 μL of chymotrypsin (total PO) or PBS (acCve PO) and with 150 μL of an L-‐ dopa soluCon were added as a substrate for the PO that ulCmately generated melanin. • The plate was placed in a Synergy HT-‐I spectrophotometer, and the absorbance was measured every two minutes at 492 nm for a total of two hours . Samples turn brown in direct proporCon to the amount of PO in them (Fig. 4).
0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 108 114 120
Time (mins)
AcCve (CirculaCng) PO AcCvity
Figure 3: Pile of termite eggs and young incipient colony where eggs and young larvae are collected from
Figure 7: Circula?ng PO is much lower than total PO. The young larvae have ~10 ?mes more PO than the eggs. Yet even Stage 2 and 3 eggs (Fig. 8) had detectable levels of ac?ve PO.
• Workers have approximately 100x more total PO acCvity than eggs per microliter and ~10x more than young larvae. • The larvae have approximately 10x more total PO acCvity than eggs per microliter • Such differences in PO acCvity are likely due to differences in the expression level of PO-‐related genes in termites as they mature • Stage 1 egg results are not shown in the results because their data produced many outliers • This inconsistent data could be caused by the vitogellin bubbles as seen in Fig. 8 • This differenCal expression of PO has never before been examined or quanCfied in such young insects • • •
Figure 4: Micro plate with samples. The darker color represents the melanin. The darker rows beneath the lighter rows represent samples that contain chymotrypsin.
References
Rosengaus, R.B., Traniello, J.F.A. 2001. Disease suscepCbility and the adapCve nature of colony demography in the dampwood termite Zootermopsis angus/collis. Behavioral Ecology and Sociobiology, 50:546–556. Tsakas, S., Marmaras, V.J. 2010. Insect immunity and its signaling: an overview. ISJ, 7: 228-‐238. Vilmos, P., Kurucz, E., 1998. Insect immunity: evoluConary roots of the mammalian innate immune system. Immunology LeWers 62: 59-‐66.
Acknowledgements
0 6 12 18 24 30 36 42 48 54 60 66 72 78 84 90 96 102 108 114 120
Time (mins)
Theo Bowe, Natalie Grace Schulz, John Paul ThoWam, Lyndie Parker, Esha Parikh, Grace Lee, Maeve Tobin, Sam Bosch. We thank the Godoy lab and Ryan Benson for the use of lab equipment and the Epstein lab for the use of the sonicator.
